It is known by those skilled in the art that nitric oxide (NO) is a biologic mediator derived from the amino acid L-arginine. A family of enzymes, known as nitric oxide synthase (NOS), act upon L-arginine to oxidize one of the guanidino nitrogens to nitric oxide while citrulline is formed from the remainder of the L-arginine molecule. Nitric oxide is a very short-lived free radical and is rapidly oxidized to nitrite (NO.sub.2.sup.-) and nitrate (NO.sub.3.sup.-) which are measured as the stable inactive end products of nitric oxide formation.
It is well known by those skilled in the art that multiple isoforms of the nitric oxide synthase enzyme exist and that they are generally classified into two broad categories: (1) constitutive and (2) inducible. These classes of NOS enzymes vary considerably in size, amino acid sequence, activity and regulation. For example, cells such as neurons and vascular endothelial cells contain constitutive NOS isoforms while macrophages and vascular smooth muscle cells express an inducible NOS.
It is generally well known that the small amounts of nitric oxide generated by a constitutive NOS appear to act as a messenger molecule by activating soluble guanylate cyclase and, thus, increasing intracellular guanosine, 3',5'-cyclic monophosphate (cGMP) and the induction of biological responses that are dependent on cGMP as a secondary messenger. For example, through this mechanism, endothelium derived nitric oxide induces relaxation of vascular smooth muscle and is identified as endothelium derived relaxing factor (EDRF) [Palmer, et al., 1987, Nature 327:524-526 and Ignarro, et al., 1987, Proc. Natl. Acad. Sci. USA 84: 9265-9269]. Another example includes, but is not limited by, neuronal nitric oxide which acts as a neurotansmitter by activating guanylate cyclase with important functions in the central nervous system and autonomic nervous system (Bredt, et al., 1989, Proc. Natl. Acad. Sci. USA 86:9030-9033 and Burnett, et al., 1992, Science 257: 401-409). It is generally known by those skilled in the art that the sustained production of nitric oxide by the inducible nitric oxide synthase has antimicrobial and antitumor functions. (see Granger, et al., 1989, J. Clin. Invest. 81: 1129-1196 and Hibbs, et al., 1987, Science 235: 479-476, respectively). It is also known by those skilled in the art that when vascular smooth muscle cells are stimulated to express a NOS enzyme by inflammatory cytokines, the large amounts of nitric oxide released contribute to the vasodilation and hypotension seen in sepsis (Busse and Mulsch, 1990, FEBS Letter 265: 133-136).
Thus, it will be appreciated that nitric oxide has normal physiologic intracellular and extracellular regulatory functions, and in some instances excessive production of nitric oxide can be detrimental. For example, stimulation of inducible nitric oxide synthesis in blood vessels by bacterial endotoxin, such as for example bacterial lipopolysaccharide (LPS), and cytokines that are elevated in sepsis results in excessive dilation of blood vessels and sustained hypotension commonly encountered with septic shock (Kilbourn, et al., 1990, Proc. Natl. Acad. Sci. USA 87: 3629-3632). It is known that overproduction of nitric oxide in lungs stimulated by immune complexes directly damages the lung (Mulligan, et al., 1992, J. Immunol. 148: 3086-3092). Induction of nitric oxide synthase in pancreatic islets impairs insulin secretion and contributes to the onset of juvenile diabetes (Corbett, et al., 1991, J. Biol. Chem. 266: 21351-21354). Production of nitric oxide in joints in immune-mediated arthritis contributes to joint destruction (McCartney, et al., 1993, J. Exp. Med. 178: 749-754).
It will be appreciated that there is a great need in the medical community for selective inhibition of the inducible form of NOS but not the constitutive types of NOS in humans because this would allow for a means of preventing, such as for example, the damage of pancreatic islets or joint destruction in arthritis without preventing the physiologic regulation of vasomotor tone or neurotransmission in the central nervous system.
In many situations nitric oxide even when produced in high amounts as seen with inducible nitric oxide synthase expression can be beneficial. For example, induced nitric oxide synthesis is important in preventing liver damage in endotoxemia (Billiar, et al., 1990, J. Leuk. Biol. 48: 565-569; Harbrecht et al, 1992, J. Leuk. Biol. 52:390-392).
Several references attempt to link nitric oxide to changes seen in vascular disease. For example, Bucala, et al. (1991, J. Clin. Inv. 87: 432-438) disclose that glycosylation products that accumulate in vessel walls during hyperglycemia may quench nitric oxide and reduce nitric oxide availability. Chin, et al. (1992, J. Clin. Inv. 89: 10-18) disclose that oxidized lipoproteins have a similar effect by inactivating nitric oxide. Chester, et al. (1990, Lancet 336: 897-900) disclose that nitric oxide synthesis is reduced in atherosclerotic epicardial arteries in humans. None of these references shed light on therapeutic avenues regarding iNOS-driven gene therapy.
Actions of nitric oxide important to vascular integrity and the prevention of the atherosclerotic lesion include vasodilation (Palmer, et al., 1987, Nature 327: 524-526; Ignarro, et al., 1987, Proc. Natl. Acad. Sci. USA 84: 9265-9269), inhibition of platelet adherence and aggregation (Radomski, et al., 1987, Br. J. Pharmacol. 92: 639-646), inhibition of vascular smooth muscle (Nunokawa, et al., 1992, Biochem. Biophys. Res. Com. 188:409-415) and fibroblast (Werner-Felmayer, et al., 1990, J. Exp. Med. 172: 1599-1607) cellular proliferation. Nitric oxide is normally produced by the vascular endothelium and, because of a very short half-life (t.sub.1/2 in seconds), diffuses only to the adjacent smooth muscle where it causes relaxation via the activation of soluble guanylate cyclase (Moncada, et al., 1991, Pharmacol. Rev. 43: 109-142). Nitric oxide released toward the lumen assists in preventing platelet adherence. L-arginine serves as the substrate for nitric oxide formation, and the small amounts of nitric oxide derived from endothelial cells is produced in an ongoing fashion (Palmer, et al., 1987, Nature 327: 524-526; Ignarro, et al., 1987, Proc. Natl. Acad. Sci. USA 84: 9265-9269) by a cNOS, which is located primarily on microsomal and plasma membranes. Agonists such as acetylcholine and bradykinin increase cNOS by activity enhancing calcium/calmodulin binding to the enzyme. The cDNA coding for this enzyme has been cloned from human endothelial cells. (Janssens, et al., 1992, J. Biol. Chem. 267: 14519-14522; Marsden, et al., 1992, FEBS Letters 307: 287-293).
We recently demonstrated that nitric oxide biosynthesis is induced in isolated human hepatocytes after stimulation with interleukin-1, tumor necrosis factor-alpha, interferon-gamma and bacterial lipopolysaccharide (bacterial endotoxin) [Nussler, et al., April 1992, FASEB J. 6(5): A1834 and Nussler, et al., 1992, J. Exp. Med. 176: 261-264)]. Heretofore no human cell type was known to show increased production of nitrogen oxides typical of iNOS expression when treated with cytokines (Drapier, 1991, Res. Immunol., Vol. 142: 557). It is generally known by those skilled in the art that all attempts to induce nitric oxide synthase in human macrophages and related cells typical to those found in rodent macrophages have failed (Drapier, Res. Immunol. 142: 562, 589-590). In spite of this background material, there remains a very real and substantial need for a cDNA clone for human tissue inducible nitric oxide synthase and a process for the molecular cloning of the same.
Inducible NOS can be expressed in cell types such as murine macrophages under conditions of cytokine and endotoxin activation (Stuehr, et al., 1985, Proc. Natl. Acad. Sci. USA 82: 7738-7742; Hibbs, et al., 1987, Science 235: 473-476; Hibbs, et al., 1987, J. Immunol. 248: 550-565), rat hepatocytes (Curran, et al., 1989, J. Exp. Med. 170: 1769-1774; Billiar, et al., 1990, Biophys. Res. Corn. 168: 1034-1040), rat vascular smooth muscle cells (Busse, et al., 1990, FEBS Letter 275: 87-90; Nakayama, et al., 1992, Am. J. Respir. Cell. Mol. Biol. 7: 471-476), and capillary endothelial cells (Gross, et al., 1991, Biochem. Biophys. Res. Com. 179: 823-829). This enzyme, which is completely absent in resting cells, produces large amounts of nitric oxide continuously over many hours. A calmodulin is tightly bound to the iNOS molecule, keeping the enzyme in a fully activated state (Cho, et al., 1992, J. Exp. Med. 176: 599-604; Iida, et al., 1992, J. Biol. Chem. 267: 25385-25388). Release of large amounts of nitric oxide after iNOS induction in macrophages has cytostatic properties and is involved in the prevention of tumor cell (Hibbs, et al., 1987, Science 235: 473-476) and microbial (Green, et al., 1992, J. Leuk. Biol. 50: 93-103) proliferation. Despite iNOS related systemic toxicity seen in various tissues, it would be advantageous to target local cell populations with a DNA sequence encoding iNOS or a biologically active fragment or derivative; such a gene therapy treatment will promote prophylactic and/or therapeutic actions in regard to diseases or disorders including but not necessarily limited to vascular occlusive disease, tumor cell growth associated with cancer, and numerous microbial infections.